TY - JOUR
T1 - Analysis of therapeutic proteins and peptides using multiangle light scattering coupled to ultra high performance liquid chromatography
AU - Espinosa-De La Garza, Carlos E.
AU - Miranda-Hernández, Mariana P.
AU - Acosta-Flores, Lilia
AU - Pérez, Néstor O.
AU - Flores-Ortiz, Luis F.
AU - Medina-Rivero, Emilio
N1 - Publisher Copyright:
© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
PY - 2015/5/1
Y1 - 2015/5/1
N2 - Analysis of the physical properties of biotherapeutic proteins is crucial throughout all the stages of their lifecycle. Herein, we used size-exclusion ultra high performance liquid chromatography coupled to multiangle light scattering and refractive index detection systems to determine the molar mass, mass-average molar mass, molar-mass dispersity and hydrodynamic radius of two monoclonal antibodies (rituximab and trastuzumab), a fusion protein (etanercept), and a synthetic copolymer (glatiramer acetate) employed as models. A customized instrument configuration was set to diminish band-broadening effects and enhance sensitivity throughout detectors. The customized configuration showed a performance improvement with respect to the high-performance liquid chromatography standard configuration, as observed by a 3 h column conditioning and a higher resolution analysis in 20 min. Analysis of the two monoclonal antibodies showed averaged values of 148.0 kDa for mass-average molar mass and 5.4 nm for hydrodynamic radius, whereas for etanercept these values were 124.2 kDa and 6.9 nm, respectively. Molar-mass dispersity was 1.000 on average for these proteins. Regarding glatiramer acetate, a molar mass range from 3 to 45 kDa and a molar-mass dispersity of 1.304 were consistent with its intrinsic peptide diversity, and its mass-average molar mass was 10.4 kDa. Overall, this method demonstrated an accurate determination of molar mass, overcoming the difficulties of size-exclusion chromatography.
AB - Analysis of the physical properties of biotherapeutic proteins is crucial throughout all the stages of their lifecycle. Herein, we used size-exclusion ultra high performance liquid chromatography coupled to multiangle light scattering and refractive index detection systems to determine the molar mass, mass-average molar mass, molar-mass dispersity and hydrodynamic radius of two monoclonal antibodies (rituximab and trastuzumab), a fusion protein (etanercept), and a synthetic copolymer (glatiramer acetate) employed as models. A customized instrument configuration was set to diminish band-broadening effects and enhance sensitivity throughout detectors. The customized configuration showed a performance improvement with respect to the high-performance liquid chromatography standard configuration, as observed by a 3 h column conditioning and a higher resolution analysis in 20 min. Analysis of the two monoclonal antibodies showed averaged values of 148.0 kDa for mass-average molar mass and 5.4 nm for hydrodynamic radius, whereas for etanercept these values were 124.2 kDa and 6.9 nm, respectively. Molar-mass dispersity was 1.000 on average for these proteins. Regarding glatiramer acetate, a molar mass range from 3 to 45 kDa and a molar-mass dispersity of 1.304 were consistent with its intrinsic peptide diversity, and its mass-average molar mass was 10.4 kDa. Overall, this method demonstrated an accurate determination of molar mass, overcoming the difficulties of size-exclusion chromatography.
KW - Biotherapeutic proteins
KW - Hydrodynamic radius
KW - Multiangle light scattering
KW - Synthetic copolymers
KW - Ultra high performance liquid chromatography
UR - http://www.scopus.com/inward/record.url?scp=84929274885&partnerID=8YFLogxK
U2 - 10.1002/jssc.201400863
DO - 10.1002/jssc.201400863
M3 - Artículo
SN - 1615-9306
VL - 38
SP - 1537
EP - 1543
JO - Journal of Separation Science
JF - Journal of Separation Science
IS - 9
ER -